Streptomyces albulus subsp. ochragerus subsp. nov. culture for making peptide

ABSTRACT

Novel peptide B-52653 having the formula: ##STR1## which is produced by cultivating a microorganism belonging to the genus Streptomyces and being capable of producing B-52653 in a culture medium, whereby B-52653 is elaborated and accumulated in the cultured broth and is recovered. 
     B-52653 is useful as a germicide or disinfectant, and a possibility of the present substance as an antifibrotic agent is suggested.

The present invention relates to a novel peptide B-52653 which isbiologically active compound and a method of producing the same.

It is well known that soil microorganisms isolated from a soil sampleproduce various biologically active compounds including antibiotics. Insearch of new biologically active compounds, the present inventorscollected a large number of soil samples, cultivated the microorganismsisolated from the samples and detected the cultures for inhibitoryactivity of cell wall synthesis and collagen-proline hydroxylase inculture broth. A new biologically active peptide was discovered in theculture broth of a microorganism. It was found that this compound notonly inhibits growth of gram-positive and gram-negative bacteria throughits cell wall synthesis inhibitory activity but also hascollagen-proline hydroxidase inhibitory activity, that saidmicroorganisms belong to the genus Streptomyces, that said peptide canbe produced and accumulated in the culture broth by cultivating saidmicroorganism in an appropriate culture medium under suitable culturalconditions. The present invention was accomplished based on theabove-mentioned findings. The present inventors named the above newpeptide B-52653. The above findings and subsequent studies have nowresulted in the present invention.

The present invention is therefore directed to (1) a novel peptideB-52653, which has the formula: ##STR2## and (2) a method of producingsaid peptide, B-52653, characterized by cultivating a B-52653-producingstrain of the genus Streptomyces to thereby cause the strain toelaborate and accumulate B-52653 in the culture broth and isolating theB-52653 from said broth.

The present invention is carried into practice using a microorganism ofthe genus Streptomyces that is able to elaborate the novel peptideB-52653. As an example of said B-52653-producing microorganisms, theremay be mentioned Streptomyces sp. strain No. B-52653 which is a strainisolated from a soil sample obtained in Akashi City, Hyogo Prefecture,Japan, by the present inventors.

In this specification, the above-mentioned novel peptide B-52653 willsometimes be referred to briefly as B-52653, and said Streptomyces sp.strain No. B-52653 as strain No. B-52653.

(A) Microbiological characteristics of Strain No. B-52653

The microbiological characteristics of Strain No. B-52653 wereinvestigated by the procedure of Schirling and Gottlieb [InternationalJournal of Systematic Bacteriology 16, 313-340, 1966]. The following arethe results of cultivation of the strain at 28° C. for 21 days.

(1) Morphological characteristics

Generally, on agar media, it gives a vegetative mycelium which is wellbranched and elongated, with its aerial mycelium being monopodiallybranched. Many of the spore chains form spiral or loop, and some areflexuous. In many instances, each spore chain is composed of more than10 spores. The spores are oval or elliptical, ranging from 0.8 to 1.2μm×1.0 to 1.5 μm with spiny surface. Neither flaggella nor sporangiawere observed.

(2) Cultural characteristics

The present strain grows well on various media, producing abundantspores, and the aerial mycelium is light gray to brownish gray. Thereverse color ranges from pale yellow to brown, and on a few kinds ofmedia, may produce light yellow to light brown soluble pigments.

(a)

Sucrose nitrate agar

Growth (G): moderate

Aerial mycelium (AM): moderate, light brownish gray to Dusty Peach(5cb-5ec)*

Soluble pigment (SP): none

(b)

Glucose asparagine agar

(G): moderate

(AM): abundant, Ashes (5fe)*

(SP): none

(c)

Glycerin asparagine agar

(G): moderate

(AM): abundant, Silber Gray (3fe)*

(SP): Lt Tan (3gc)*

(d)

Starch agar

(G): moderate

(AM): abundant, Silver Gray (3fe)*

(SP): none

(e)

Tyrosine agar

(G): moderate

(AM): moderate, Lt Ivory (2ca)* to Pussywillow Gray (5dc)*

(SP): Rose Taupe (5ig)*

(f)

Nutrient agar

(G): moderate

(AM): moderate, white to Pearl (3ba)*

(SP): none

(g)

Yeast extract malt extract agar

(G): abundant

(AM): abundant, Beaver (4li)* to Lead Gray (5ih)*

(SP): none, or pale yellow-brown

(h)

Oatmeal agar

(G): abundant

(AM): abundant, Silver Gray (3fe)*

(SP): Honey Gold (2ic)*

(i)

Calcium malate agar

(G): moderate

(AM): moderate, Bisque (3ec)*, to white

(SP): Lt Fawn (4ge)*

(3) Physiological characteristics

(a) Temperature range for growth: 9°-40° C.

(b) Liquefaction of gelatin (glucose peptone gelatine): positive (weak)

(c) Hydrolysis of starch: positive

(d) Coagulation of skimmed milk: peptonized

(e) Production of melanoid pigments: Tyrosine agar: false positivePeptone yeast agar: negative

(4) Assimilation of carbon sources

    ______________________________________                                        L-arabinose                                                                              ±        D-xylose ±                                          D-glucose  +++         D-fructose                                                                             +++                                           Sucrose    ++          Inositol +++                                           L-rhamnose +           Raffinose                                                                              ±                                          D-mannitol +++         Control  ±                                          ______________________________________                                         (Note)                                                                        +++: abundant growth                                                          ++: good growth                                                               +: growth                                                                     ±: slight growth                                                      

As to the above-mentioned characteristics of strain No. B-52653,reference was made to S. A. Waksman: The Actinomycetes, Vol. 2 (TheWilliams and Wilkins Co., 1961); R. E. Buchanan and N. E. Gibbons (ed.),Bergy's Manual of Determinative Bacteriology, 8th Edition, 1974;International Journal of Systematic Bacteriology Vol. 18, No. 2, pp.69-189 and No. 4, pp. 279-392 (1968), ditto Vol. 19, No. 4, pp. 391-512(1969) and ditto Vol. 22, No. 4, pp. 265-394 (1972), and otherliterature.

The taxonomic position of this strain based on the above-mentionedcharacteristics thereof is that it apparently belongs to the sectionSpirales or Retinacuriapelti and the Gray series as proposed by Pridhamet al (Applied Microbiology 6, 52-79, 1958). As a known species whichseems to be most closely related to the present strain, there may bementioned Streptomyces albulus. Accordingly, a comparison was made ofstrain No. B-52653 with Streptomyces albulus IFO 13410 (ISP 5492).Strain No. B-52653 produces a light brown-gray aerial mycelium onsucrose nitrate agar and a light yellow-gray aerial mycelium on glucosenitrate agar. When grown on calcium malate agar, the same strainproduces a light brown soluble pigment. Moreover, it grows utilizingL-rhamnose and sucrose. On the other hand, Streptomyces albulus producesa white aerial mycelium on sucrose nitrate agar and glucose nitrate agarand does not produce a soluble pigment on calcium malate agar. Moreover,the latter strain does not assimilate L-rhamnose or sucrose and,therefore, does not grow on these carbon sources.

The above results suggested that strain No. B-52653 was a novelsubspecies of Streptomyces albulus and, accordingly, the strain wasproposed the name Streptomyces albulus subsp. ochragerus subsp. nov.

This strain No. B-52653 has been deposited in Fermentation ResearchInstitute, the Agency of Industrial Science and Technology (FERM),Ibaragi, Japan under the deposit number of FERM BP-273, Institute forFermentation, Osaka (IFO), Japan under the accession number of IFO14072, and The American Type Culture Collection (ATCC), U.S.A. under theaccession number of ATCC 31713.

While, as mentioned above, strain No. B-52653 is a new strain of thegenus Streptomyces, and it may undergo variation and mutation, as ageneral trait of microorganisms, either spontaneously or under theinfluence of mutagen. For example, mutants of the strain can be obtainedby means of X-rays, gamma-rays, ultraviolet light or other radiation,monospore separation, treatment with various chemicals, cultivation onmedia containing such chemicals, and so forth. These mutants, as well asspontaneous mutants, can also be employed for the purposes of thepresent invention unless they are substantially considered to be newspecies in view of the above-mentioned or undermentioned microbiologicalcharacteristics and as long as they are capable of elaborating B-52653.By way of example, when strain No. B-52653 is subjected to variousmutagenic treatments, there are obtained mutants which produce yellow orblue aerial mycelia.

The culture medium used for the cultivation of this invention may beeither liquid or solid, only if it contains nutrients which the strainemployed is capable of utilizing. However, when mass production iscontemplated, a liquid medium is more advantageous. In the medium areincorporated the carbon and nitrogen sources which the strain canassimilate and digest, inorganic materials, and trace nutrients. Thecarbon sources may include, for example, glucose, lactose, sucrose,maltose, dextrin, starch, glycerin, mannitol, sorbitol, oils and fats(e.g. soybean oil, lard oil, chicken oil, etc.), and the nitrogensources may include, among others, meat extract, yeast extract, driedyeast, soybean flour, corn starch liquor, peptone, cottonseed flour,spent molasses, urea, ammonium salts (e.g. ammonium sulfate, ammoniumchloride, ammonium nitrate, ammonium acetate, etc.) and so on. Inaddition, there are incorporated suitable amounts of salts of sodium,potassium, calcium, magnesium, etc.; salts of iron, manganese, zinc,cobalt, nickel, etc., salts of phosphoric acid, boric acid, etc., andsalts of organic acids such as acetic acid, propionic acid, etc. Theremay also be added amino acids (e.g. glutamic acid, aspartic acid,alanine, lysine, valine, methionine, proline, etc.), peptides (e.g.dipeptides, tripeptides, etc.), vitamins (e.g. B₁, B₂, nicotinic acid,B₁₂, C, etc.), nucleic acids and related compounds (e.g. purine orpyrimidine derivatives). Of course, for the purpose of adjusting the pHof the medium, inorganic or organic acids, alkalis, buffers, etc. may beadded to the medium. Suitable amounts of oils, surfactants, etc. mayalso be added for defoaming purposes.

The cultivation may be carried out by stationary culture, shakingculture, submerged culture or other known cultural procedure. For massfermentation, the procedure of so-called submerged culture is of courseadvantageous. While, of course, the cultural conditions depend on thecondition and composition of medium, particular strain used, culturalmethod, etc., cultivation is usually conducted at a temperature of 20°to 35° C. and with an initial pH in the range of pH about 5 to 8.Preferred conditions are 23° to 32° C. and pH 5.5 to 7.0 (initial).While the cultivation time is also dependent on the above-mentionedconditions, cultivation is preferably continued until the amount of thepresent peptide produced reaches maximal. The time required forattaining such a concentration maximum is normally about 2 to 8 days inthe case of shaking or aerated stirring culture using a liquid medium.

The pH suited to the production of the present peptide is weakly acidic,the optimum range being pH 5.5 to 7.0, and the output of the substancecan be increased by controlling the pH of the medium with an acid oralkali. Thus, the yield of the substance can be considerably increasedby adding inorganic phosphates in a concentration of 50 to 1000 ppm tothe medium or by using saccharides such as glucose, starch or/andsucrose as carbon sources together with selected nitrogen sources andinorganic salts.

While its proportion varies with cultural conditions, B-52653 is usuallyaccumulated extracellularly, with about 10% of the total yield beingsometimes detected in the cells.

The present peptide thus produced in the culture broth can be assayed bythe agar-well method or paper disk method using a drug-resistant mutantof Staphylococcus aureus as the test organism and B-52653 as thereference standard. Medium A [glucose 3%, sodium glutamate 0.5%, K₂ HPO₄0.05%, MgSO₄.7H₂ O 0.05%, KCl 0.05%, yeast extract (Difco) 0.05%,Casamino acid (Difco) 0.02%, agar (Difco)].

The present peptide is mostly produced in the filtrate of the culturebroth. The B-52653 thus produced can be isolated by the proceduresconventionally used for harvesting microbial metabolites from culturebroths, either as used singly or in a combination or as applied inrepetition. Thus, for example, there may be employed such procedures asfiltration, centrifugation, dialysis, concentration, drying,freeze-drying, adsorption and desorption, means utilizing a differencein solubility in different solvents (e.g. precipitation,crystallization, recrystallization, extraction counter-currentdistribution, etc.), chromatography, etc.

More particularly, since B-52653 is produced and accumulatedextracellularly for the most part, this peptide is preferably recoveredfrom the liquid fraction of the culture broth after removal of thecellular fraction. Moreover, because this peptide is an amphotericwater-soluble compound having carboxyl and amino functions, it can beadvantageously isolated and purified from the liquid fraction of thebroth by taking advantage of this property.

The chromatography referred to above may be advantageously conductedusing cation and anion exchangers (e.g. ion exchange resins, Sephadexion exchangers, ion exchange cellulose, etc.), gel permeation carriers[e.g. Sephadex (Pharmacia Fine Chemicals, Sweden), Biogel (Bio-RadLaboratories, U.S.A.), preferably Sephadex LH-20 Pharmacia FineChemicals, Sweden], activated carbon, high-porous polymers [e.g.preferably Amberlite XAD-2 (Rohm and Haas Co., U.S.A.), Diaion HP-10(Mitsubishi Kasei Kogyo, Japan)], alumina, florisil, silica gel,cellulose and so on.

The present invention isolated and purified the present compound by theabove-mentioned procedures, investigated its physicochemical properties,and also through an independent research, found that it is a newcompound of the following structural formula: ##STR3##

B-52653 can be produced in the above manner. Its physicochemicalproperties, as measured with the samples produced in accordance withExamples 1 and 2, are as follows.

(1) White powder

(2) Elemental analysis: Calcd. for C₉ H₁₅ N₃ O₅.H₂ O: C, 38.43; H, 6.81;N, 14.94, Found: C, 38.44; H, 6.34; N, 14.78.

(3) Molecular weight (neutralization equivalent) and pKa'

The neutralization equivalent determined from the dissociation curve(titration in an aqueous solution containing 0.1N--HCl with 0.1N--NaOH)is 140±10, and the pH of the solution at the half-equivalent pointdetermined from the dissociation curve is Ph 8.3±0.5. This dissociationcurve is considered to be a composite of the dissociation curves of twodissociable groups having pKa' values close to each other (presumably atabout 7.6 and about 8.8, although accurate pKa' values could not bemeasured because of the absence of an inflection point). Therefore, themolecular weight of the present peptide was assumed to be 280±20. When1N--HCl was added to an aqueous solution of this substance and titrationwas performed with 1N--NaOH as aforesaid, a dissociable group having apKa' value at about 3.15 was also detected.

(4) Optical rotation

    [α].sub.D.sup.25 +50°±5° (c=1.0, H.sub.2 O)

    [α].sub.D.sup.25 +62°±5° (c=1.0, 0.1N HCl)

    [α].sub.D.sup.25 +50°±5° (c=1.0, 0.1N NaOH)

(5) Ultraviolet absorption spectrum

The ultraviolet absorption spectrum of the present substance in aqueoussolution shows no characteristic absorption maximum, except endabsorptions, between 220 and 360 nm.

(6) Infrared absorption spectrum

The wave-numbers (cm⁻¹) of main absorption peaks in the infraredspectrum (KBr) are as follows. P 3400, 1690, 1610, 1395, 1265, 1205,1080

(7) Nuclear magnetic resonance spectrum

The following nuclear magnetic resonance spectrum was observed indeuterium oxide using a Varian EM-390 (90 MHz) spectrometer.

δ: 1.70 (3H, d, J=7); 1.75 (m), 2.26 (m), 2.78 (m) (total 2H); 3.24 (1H,m), 4.26 (1H, q, J=7), 4.71 (1H, d, J=4.5), 5.38 (1H, m).

(8) Color reactions

Ninhydrin reaction: positive

Greig-Leaback peptide reaction: positive

Sakaguchi reaction: negative

Anthrone-sulfuric acid reaction: negative

Orcin-sulfuric acid reaction: negative

(9) Solubility

Readily soluble in water, but either only sparingly soluble or insolublein organic solvents such as methanol, ethanol, acetone, chloroform,ethyl acetate, benzene, ethyl ether, petroleum ether, pyridine, glacialacetic acid, dimethylformamide, dimethyl sulfoxide, etc.

(10) Thin-layer chromatography (Merck's precoated TLC plate, silica gel60F-254)

    ______________________________________                                        Solvent systems           Rf                                                  ______________________________________                                        1-Butanol-acetic acid-water                                                                             0.15                                                (3:1:1)                                                                       1-Butanol-pyridine-acetic acid-water                                                                    0.08                                                (4:1:1:2)                                                                     1-Propanol-water          0.13                                                (7:3)                                                                         2-Propanol-diisopropyl ether-                                                                           0.16                                                60% formic acid                                                               (4:3:3)                                                                       Chloroform-methanol-17% aqueous ammonia                                                                 0.17                                                (2:2:1)                                                                       ______________________________________                                    

Antimicrobial activity

In the assays employing bacteria as test organisms, a loopful of 10⁶ CFU(cell forming unit)/ml was used as the inoculum and the MIC's weredetermined after incubation at 37° C. for 18-20 hours. In the case offungi and yeasts, a loopful of each microorganism was milled thoroughly,a loopful of the resultant suspension was inoculated, and the MIC wasdetermined after incubation at 28° C. for 2 or 3 days. The measurementof MIC was carried out by the agar dilution method using the media shownin the table.

It will be apparent from the table that B-52653 displays strong activityagainst gram-positive and gram-negative bacteria and that this substanceis as active against various antibiotic-resistant microorganisms asagainst the corresponding susceptible strains. It has also been shownthat the present substance displays strong activity against certainphytopathogenic bacteria.

    ______________________________________                                                                       MIC                                            Test organism         Medium   (μg/ml)                                     ______________________________________                                        Staphylococcus aureus IFO 13276                                                                     I        0.39                                           Staphylococcus epidermidis FS 5010                                                                  I        0.39                                           Bacillus subtilis PCI 219                                                                           II       6.25                                           Bacillus cereus IFO 3001                                                                            II       3.13                                           Bacillus megaterium IFO 3970                                                                        II       0.1                                            Escherichia coli NIHJ JC-2                                                                          II       3.13                                           Escherichia coli 0-111                                                                              II       6.25                                           Escherichia coli K-12 W3110                                                                         II       3.13                                           Escherichia coli TN 647                                                                             II       3.13                                           Proteus vulgaris IFO 3988                                                                           II       1.50                                           Proteus mirabilis IFO 3847                                                                          II       1.56                                           Proteus morganii IFO 3168                                                                           II       3.13                                           Klebsiella pneumoniae IFO 3512                                                                      II       12.5                                           Klebsiella pneumoniae GN 3848                                                                       II       6.25                                           Citrobacter freundii TN 457                                                                         II       1.56                                           Citrobacter freundii TN 564                                                                         II       3.13                                           Enterobacter cloacas IFO 12937                                                                      II       25                                             Enterobacter aerogenes TN 582                                                                       II       6.25                                           Salmonella typhimurium LT-2                                                                         II       0.78                                           Salmonella enteritidis IFO 3313                                                                     II       0.2                                            Serratia marcescens  IFO 12648                                                                      II       0.78                                           Serratia marcescens TN 24                                                                           II       0.2                                            Pseudomonas aeruginosa IFO 3080                                                                     II       >100                                           Pseudomonas aeruginosa J-31                                                                         II       >100                                           Alternaria kikuchiana IFO 7515                                                                      III      >100                                           Pyricularia oryzae KHG-1                                                                            III      10                                             Cochlioborus miyabeanus IFO 5277                                                                    III      100                                            Botrytis cinerea TKF-12                                                                             III      50                                             Sclerotinia sclerotiorum IFO 9395                                                                   III      1                                              Pellicularis sasakii KHG-2                                                                          III      20                                             Penicillium chrysogenum IFO 4626                                                                    IV       >100                                           Aspergillus niger IFO 4066                                                                          IV       >100                                           Saccharomyces cerevisiae IFO 0209                                                                   IV       12.5                                           Candida albicans IFO 0538                                                                           IV       >100                                           ______________________________________                                    

Medium I: 3% glucose, 0.5% sodium glutamate, 0.05% K₂ HPO₄, 0.05%MgSO₄.7H₂ O, 0.05% KCl, 0.05% yeast extract (Difco), 0.02% Casaminoacid, 1.5% agar (pH 7.0).

Medium II: 0.5% glucose, 0.01% yeast extract, 0.1% (NH₄)₂ SO₄, 0.01%MgSO₄.7H₂ O, 0.05% sodium citrate, 0.2% KH₂ PO₄, 0.7% K₂ HPO₄, 1.5% agar(pH 7.0) [F. R. Atherton et al. Antimicrobial Agents and Chemotherapy15, No. 5, 677-683 (1979)].

Medium III: potato sucrose agar

Medium IV: Peffer's modified agar medium: 3.0% sucrose, 0.2%L-asparagine, 0.3% NH₄ NO₃, 0.1% KH₂ PO₄, 0.1% MgSO₄.7H₂ O, 0.001%Versenol*, agar 1.5% (pH 7.0) [* iron sodium ethanolethylenediaminetriacetate 50%].

The medium IV was supplemented with the following substances (per 100ml) before use: 100 μg vitamin B₁ hydrochloride, 100 μg vitamin B₂, 100μg calcium pantotheate, 100 μg nicotinic amide, 0.5 μg biotin, 50 μgfolic acid, 200 μg vitamin B₆ hydrochloride, 50 μg p-aminobenzoic acid,0.2 μg vitamin B₁₂.

Anti-infective effects in mice

Male mice of the ICR/SLC strain, 4 weeks of age, were intraperiotoneallyinfected with 2×10⁸ CFU cells from an overnight culture ofStaphylococcus aureus E-97 in Brain heart infusion broth (Difco).Immediately thereafter, an aqueous solution of B-52653 wasintraperitoneally administered in a single dose. As shown below, theabove treatment had a prophylactic effect against infection.

    ______________________________________                                        Dosage (mg/kg)                                                                3.13    6.25   12.5    25   50   100  ED.sub.50                                                                           (mg/kg)                           ______________________________________                                        S/T  1/5    2/5    2/5   3/5  3/5  5/5        17.7                            ______________________________________                                         *S: the number of surviving animals                                           T: the number of mice submitted to the test                              

Collagen proline hydroxylase inhibitory activity

Organ fibrosis, inclusive of hepatic cirrhosis and pulmonary fibrosis,has been considered to be a disease caused by a pathological overgrowthof collagen, and an inhibitor of the above enzyme is thought to be ofuse for suppressing such fibrosis.

The collagen prolyl hydroxylase inhibitory activity of B-52653 wasassayed by the method of R. E. Rhoads et al. [(Methods in EnzymologyXVII B, 306 (1971)]. The assay revealed that this substance causes a 50%inhibition at the concentration of 270 μg/ml. Then, the collagenbiosynthesis inhibitory action of B-52653 in rats was investigated bythe B method of Ishimaru et al. (Ishimaru T., Kanamaru, T. and Okazaki,H., the Proceedings of the 1979 Congress of the Japanese Society ofAgricultural Chemistry, p. 373). The results were that the collagensynthesis inhibition rates in rats given 50 mg/kg, 100 mg/kg and 200mg/kg were 30%, 43% and 55%, respectively.

Thus, a possibility of the present invention substance as anantifibrotic agent is suggested.

Toxicity

In acute toxicity tests in which B-52653 was administered to mice by theintravenous and intraperitoneal routes, no death occurred even at thehigh dose of 1000 mg/kg, nor was observed any particular abnormalityduring a 7-day observation period following the administration or onautopsy. It is, therefore, considered that B-52653 is a very sparinglytoxic substance.

As described above in detail, the present substances B-52653 hasbiological activities such as antimicrobial activity and activity tosuppress fibrosis of animal tissues.

The present substance in the form of an aqueous solution from about 10to about 100 μg/ml can be used as a disinfectant for bird cages,laboratory equipment, human hands, etc.

Because the present substance has activity to inhibit collagen prolylhydroxylase activity, it is of value as a reagent for a study of themechanism of collagen biosynthesis. It is also a promising applicationfor the present substance to aseptically dissolve this substance insterile physiological saline at the rate of about 2 to 10 g per 20 to100 ml/day and administer the solution to humans by intravenous dripinfusion for inhibition of the progress of hepatic fibrosis.

The following examples are further illustrative of this invention.

EXAMPLE 1

Strain No. B-52653 (IFO 14072, ATCC 31713) was inoculated on glucoseasparagine agar and incubated at 24° C. for 10 days. The grown sporeswere scraped into sterile water to prepare a spore suspension (viablecells: 3×10⁸ /ml), which was stored in a refrigerator for use as aninoculum. One milliliter of the inoculum was inoculated a 2-literSakaguchi flask containing 500 ml of a sterilized preculture medium (35g corn steep liquor, 10 g Proflo, 1 g K₂ HPO₄, 15 g CaCO₃, 20 g glucose,1 l tap water; pH 6.5), and the flask was incubated on a reciprocatingshaker at 28° C. for 40 hours. A 500 ml portion of the resultant culturewas transferred to a 200-liter stainless steel fermentor containing 100l of a sterilized preculture medium similar to the above, andcultivation was carried out at 28° C. for 24 hours with aeration (50l/min.) and stirring (200 r.p.m., 1/2 DT), and at an internal pressureof 1 kg/cm². The resulted seed culture (100 l) was further transferredto a 2000-liter stainless steel fermenter containing 900 l of asterilized main fermentation medium consisting of 500 g DL-alanine, 1 kgDL-methionine, 1 kg FeSO₄, 500 g ZnSO₄, 200 g MgSO₄.7H₂ O, 100 g MnSO₄,1.3 kg KH₂ PO₄, 25 kg Proflo, 5 kg soybean flour, b 5 kg NH₄ Cl, 12.5 kgCaCO₃ and 100 kg glucose [separately sterilized]). The fermentation wascarried out at 28° C. for 54 hours with aeration (1000 l/min.) andstirring (200 r.p.m., 1/3 DT-2 stages), and at an internal pressure of 1kg/cm². As a result, 254 μg/ml of B-52653 was produced in the broth.

EXAMPLE 2

To 980 l of the culture broth obtained in Example 1 was added 30 kg ofTopcolite No. 34 (Toko Perlite Kogyo, Japan) as a filter aid, and themixture was filtered by using a continuous vacuum filter. To 1350 l ofthe resulting filtrate was added 1.4 kg of oxalic acid and, afterstirring for 30 minutes, 5 kg of Topcolite was added, followed byfiltration. The resulting filtrate (1450 ml) was adsorbed on a column(350 l) of Amberlite 200 (H⁺ -form) and, after washing with water,elution was carried out with 0.5N-aqueous ammonia. The active fractionswere collected and concentrated under reduced pressure. The concentrate(20 l) was adsorbed on a column (200 l) of activated carbon (Shirasagifor Chromatography, Takeda Chemical Industries, Ltd., Japan) and elutionwas carried out with water. The active fractions were combined andconcentrated to 1.5 liters.

A 750 ml portion of the concentrate was adsorbed on a column (3 l) ofalumina (Merck, West Germany activated alumina 90, neutral, activity I)and, after washing with water, elution was carried out with 0.2N-aqueousammonia. The active fractions were collected and concentrated to drynessunder reduced pressure. The residue (about 30 g) was dissolved in 60 mlof water, the solution was chromatographed on activated carbon (1 l),and elution was carried out with water. The active fractions werecollected and concentrated under reduced pressure. The concentrate wasfurther adsorbed on a column (700 ml) of Amberlite IRA 68 (Rohm and HaasCo. U.S.A.), and after washing with water and with 0.1M acetic acid,elution was carried out with 0.2M acetic acid. The active fractions werecollected and concentrated under reduced pressure. The concentrate wasadsorbed on a column (500 ml) of activated carbon and eluted with water.The active fractions were combined and concentrated under reducedpessure to give 3.9 g of white powder (purity: about 93%).

The above white powder (1.0 g) was dissolved in 4 ml of 50% aqueousmethanol, diluted with 4 ml of 70% methanol and subjected to columnchromatography on Sephadex LH-20 (Pharmacia Fine Chemicals, Sweden;swollen with 70% aqueous methanol). Elution was carried out with 70%aqueous methanol. The active fractions were pooled and the methanol wasdistilled off under reduced pressure. The resulting aqueous solution wasfreeze-dried to give 0.9 g of the desired B-52653.

What we claim is:
 1. A biologically pure culture of the microorganismbelonging to the genus Streptomyces having the characteristicsidentifiable with ATCC 31713, said culture being capable of producing ina culture medium containing assimilable carbon and digestible nitrogensources, a recoverable amount of peptide B-52653 of the formula ##STR4##2. A method for producing peptide B-52653 of the formula ##STR5## whichcomprises cultivating Streptomyces albulus subsp. ochragerus subsp. nov.(ATCC 31713) in a culture medium containing assimilable carbon sourcesand digestible nitrogen sources until peptide B-52653 is substantiallyaccumulated in the culture broth, and isolating the same.